We are committed to the in-depth analysis of microbial virulence strategies using as a model organism the bacterium Legionella pneumophila, the causative agent of a potentially fatal respiratory infection known as Legionnaires' disease. The number of Legionnaires' disease cases in the U.S. has increased four-fold over the past 15 years, making L. pneumophila a significant health threat and a considerable economic burden. Like many other microbial pathogens, L. pneumophila have developed a variety of strategies to infect their human host and cause disease. They use type IV secretion system (T4SS) to deliver bacterial proteins, called effectors, into host cells. The effectors help to modulate signaling events within the host in order to create conditions favorable for L. pneumophila survival. Obtaining a detailed understanding of Legionella's biology and its virulence strategies is essential to more effectively prevent, diagnose, and treat this dangerous pneumonia, and will profoundly improve people's lives and wellbeing. L. pneumophila is ubiquitously found in freshwater habitats such as cooling towers, faucets and shower heads, or water fountains. Major outbreaks of Legionnaires' disease occur when water from contaminated sources is aerosolized and subsequently inhaled by humans. Immune-compromised individuals, infants, or the elderly are at an elevated risk of contracting an infection. According to the Center for Disease Control and Prevention (CDC), the number of diagnosed Legionnaires' disease cases within the U.S. has tripled over the past decade and a half, making this microorganism an emerging public health threat. Upon inhalation, L. pneumophila infects and replicates within alveolar macrophages, specialized immune cells within the human lung. L. pneumophila delivers close to 300 proteins, called effectors, through a T4SS into the host cell. The combined activity of the effectors allows L. pneumophila to escape the degradative endo-lysosomal pathway and, instead, to establish a camouflaged replication compartment that supports L. pneumophila growth. Most L. pneumophila effector proteins have not been characterized in detail, and their activities and host targets remain unknown. Interference with T4SS activity renders L. pneumophila avirulent, underscoring the important role of the translocated effectors for infection. The infection cycle of L. pneumophila shows numerous parallels to the virulence programs of Salmonella, Chlamydia, Mycobacterium, Coxiella, and many other human pathogens that manipulate host cells from within a membrane-enclosed compartment. In addition, given that a type IV secretion system (T4SS), the major virulence apparatus of L. pneumophila, is present in numerous animal and plant pathogens including Helicobacter or Agrobacterium, the in-depth analysis of this translocation system and its cargo proteins, called effectors, is of great importance for our general understanding of microbial virulence. Last but not least, the effector proteins that are used by L. pneumophila to manipulate host cell processes display remarkable parallels to eukaryotic proteins, and deciphering their function will yield valuable insight into mechanistic and regulatory concepts about processes that occur within our own cells. Hence, a major focus of our research is to obtain in-depth insight into their biological functions. Over the past funding period, we have developed and successfully implemented new research tools that allowed us to decipher the secrets of L. pneumophila virulence. We have developed a human proteome-based platform for the identification of human targets of L. pneumophila effectors. This platform uses a commercially available protein microarray that contains more than 9,000 human proteins. Upon incubation of the array with a fluorescently labeled effector, binding to immobilized human proteins can be detected directly on the array using a microplate reader. We also adapted this platform for the detection of posttranslational modifications. such as phosphorylation. L. pneumophila encodes several effectors with homology to eukaryotic protein kinases. By incubating the array with an effector kinase and a fluorophore-conjugated ATP analog, putative phosphorylated targets can be directly visualized directly on the array. This platform formed the basis for the study of a variety of L. pneumophila effectors, including those with kinase activity. In one project, we discovered that the L. pneumophila effector kinase LegK7 directly phosphorylates human MOB1, a scaffold protein of the Hippo pathway. The Hippo pathway is highly conserved in eukaryotes and well known for its role in controlling cell development but also tumorigenesis. Yet, the fact that a Legionella effector targets this pathway suggested an additional role of Hippo signaling in immunity. We found that LegK7 combines functional features of not just one but two separate kinases of the Hippo pathway, thereby taking control of this signaling route and causing changes in host cell gene expression that promote intracellular bacterial growth. Interference with the change in gene expression rendered human cells less susceptible to L. pneumophila growth, providing us with a new way to treat infections by this pathogen. This is a fascinating example of molecular mimicry where a Legionella effector hijacks a highly conserved eukaryotic signaling pathway. Our findings also underscore the fact that studying intracellular pathogens can teach us important lessons on how our very own cells function, thus holding the key to obtaining in-depth insight into congenital and acquired human diseases, including cancer.